Name: optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue
Uploaded: 2018-06-14T14:30:00
Description: Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

We are grateful to the donors and their families who made this work possible. We also appreciate the assistance of staff at the International Institute of Medicine and Tennessee Donor Services for helping coordinate collection of tissue used in this study. This work was supported by grants from the National Institutes of Health, NIH OT2-OD023850 to EMS2 and stipend support from NIH T32-DK007673 to AMZ. We are grateful to staff of the Vanderbilt Translational Pathology Shared Resource for access to the LCM Instrument and advice on tissue preparation. The Vanderbilt Tissue Pathology Shared Resource is supported in part by NIH grants P30-CA068485-14 and U24-DK059637-13. We thank the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine for help with genomic analysis. The GTAC is partially supported by NCI Cancer Center Support Grant #P30 CA91842 to the Siteman Cancer Center and by ICTS/CTSA Grant #UL1TR002345 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of NCRR or NIH.

All protocols described here have been approved by the Vanderbilt University Institutional Review Board (IRB).
1. Preparation Prior to Tissue Arrival
<ol>
	<li>Obtain proper IRB approval and coordinate with human organ donation agencies to obtain freshly-resected, unfixed intestinal tissue from donors that meet all research criteria required for the study. 
	&#8203;Note: This protocol can be adapted for use with mice and other rodent models.
	<ol>
		<li>Immediately upon resection of eac...

<ol>
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This procedure enables the efficient collection of numerous enteric ganglia as a source to derive RNA for RNA-seq. Here, we have accelerated the processes outlined in existing protocols while maximally preserving RNA integrity. As all steps in this procedure are interdependent, it is important that all issues be eliminated from the onset of the study and that all samples are prepared as similarly to one another as possible, to obtain reliable RNA-seq.
From the onset of the procedure, RNA integ...

This method is designed to obtain high-quality RNA samples of enteric ganglia from human intestinal tissue using laser capture microdissection (LCM). The protocol described here has been optimized to provide sufficient RNA quality and yields for RNA sequencing (RNA-seq) and is intended to be used with freshly-resected, unfixed, flash-frozen human intestinal tissue.
Functional gastrointestinal and gut motility disorders affect one of every four people in the United States. The enteric nervous system (ENS), also referred to as the second brain1, is often at the center of these disorders, as it plays a crucial role in gut homeostasis and motility. Manipulation of gut motility has generally been restricted to surgical resection of the aganglionic/noncontractile tissue, chronic dietary modification and/or medications. Surprisingly, the full transcriptome of the adult ENS remains to be sequenced, greatly limiting our ability to identify molecules within the ENS that can be targeted pharmaceutically or utilized in stem cell therapies.
There are relatively few methods for isolating RNA from human enteric ganglia. The first approach, cell dissociation2, requires high incubation temperatures and long incubation times; both of which are known to promote RNA degradation and alter the transcriptome2,3. An alternative approach, LCM, more reliably preserves the transcriptome and protects RNA integrity. Although several studies have used LCM to collect ganglia from fresh-frozen human intestinal tissue4,5,6, these approaches were either hampered by poor RNA quality and quantity, were quite labor-intensive, or needed modification of staining or RNA extraction techniques to work in our hands. Other LCM protocols designed for preserving RNA that were found in LCM product manuals provided additional improvements7,8, but adaptation was needed when applied to the isolation of enteric ganglia8,9. For these reasons, we developed an optimized protocol based on these resources that yields substantial quantities of high-integrity RNA from human enteric ganglia, has a relatively fast workflow, and produces consistent results across a large number of samples.
In this study, we present a synopsis of optimized procedures that facilitate the isolation of high-integrity RNA from enteric ganglia sourced from resected human intestinal tissue. Our method incorporates five important aspects. First, freshly-resected, unfixed human intestinal samples should be trimmed to size, have all excess moisture removed with a laboratory tissue and flattened in a large base mold before flash-freezing atop a slurry of dry ice and 2-methylbutane (2-MB). Second, histologic sections of intestine should be prepared to obtain the full plane of the myenteric plexus on a slide, which offers a large payload of enteric ganglia. Success with this step is largely dependent on the tissue preparation process. Third, the nonuniform structure of ganglia in the ENS requires the use of polyethylene napthalate (PEN) membrane slides6, which offer the greatest speed and precision during the LCM process. Fourth, ethanol-compatible dyes, such as Cresyl Violet, should be used to preserve RNA integrity while staining enteric ganglia. Last, the RNA extraction process is critical for a successful outcome with RNA-Seq. We sought an RNA extraction approach that produces high RNA integrity, maximizes RNA yields when starting with small collections of enteric ganglia, eliminates DNA contamination, and retains as many RNA species as possible.
Taken together, optimization of these factors in the present study greatly accelerates the workflow and yields samples of enteric ganglia with exceptional RNA quantity and quality. Results have been largely consistent among a sizable group of samples, indicating the consistency of this approach. Further, we have used these approaches to successfully sequence dozens of RNA samples from enteric ganglia. The strategies highlighted here can also be broadly adapted for performing LCM of desired ganglia or nuclei of the peripheral and central nervous system and other cases requiring the isolation of high-quality RNA.

<table border="0" cellpadding="0" cellspacing="0" style="width:920px;" width="919">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">10% Neutral-buffered formalin</td>
			<td style="width:280px;">Sigma</td>
			<td style="width:131px;">HT501128-4L</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Methylbutane</td>
			<td>Fisher</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mL syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mL conical tubes, polypropylene&#160;</td>
			<td>Corning</td>
			<td>05-526B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Base molds 37x24x5mm, disposable</td>
			<td>Electron Microscopy Sciences</td>
			<td>5025511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Belzer UW&#160; Cold Storage Solution</td>
			<td>Bridge to Life</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CapSure Macro LCM caps</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cling Wrap, Press&#8217;n Seal&#160;&#160;</td>
			<td>&#160;Glad&#160;&#160;</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cresyl Violet Acetate</td>
			<td>Acros Organics</td>
			<td>AC405760025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting Board</td>
			<td>Electron Microscopy Sciences</td>
			<td>63308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 190-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 200-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Microscope slides</td>
			<td>Fisher</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gloves, extended cuff</td>
			<td>Microflex&#160;</td>
			<td>19010144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gowns, surgical-disposable</td>
			<td>Kimberly-Clark&#160;</td>
			<td>19-088-2116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tray, 20 L (large polypropylene sterilizing pan)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>1335920B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (large)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (small)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Capture Microdissection System (ArcturusXT)</td>
			<td>ThermoFisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Cryostat Chuck, large,&#160; 40 mm&#160;</td>
			<td>Southeast Pathology Instrument Service</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Microscope</td>
			<td>Olympus</td>
			<td>CX43</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (20X)</td>
			<td>Olympus UPlanFl 20X/0.50, &#8734;/0.17</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (10X)</td>
			<td>Olympus UPlanFl 10X/0.25, &#8734;/-</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Sieves</td>
			<td>Acros Organics</td>
			<td>AC197255000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PicoPure RNA extraction kit</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pellet Pestle</td>
			<td>Kimble Kontes</td>
			<td>4621973</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEN membrane LCM slides</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free tubes, 1.5 mL</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZAP&#160;</td>
			<td>Sigma</td>
			<td>Sigma-R2020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;A&#34; (PicoPure)</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;B&#34; (RNeasy&#160; Micro kit)</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Method &#34;C&#34; (TRIzol)</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;D&#34; (RNeasy PLUS Mini kit)</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splash Shield, disposable faceshield</td>
			<td>Fisher</td>
			<td>17-310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors, Surgical, 14.5 cm &#34;sharp-sharp&#34;</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, cellulose acetate (0.2 &#956;m)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>190-2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Freezing Medium</td>
			<td>General Data Healthcare</td>
			<td>TFM-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylenes</td>
			<td>Fisher</td>
			<td>X3P1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

We have made several improvements to existing protocols that enable the relatively rapid collection of enteric ganglia from human intestinal samples using LCM, meeting the standards for RNA-Seq. First, we optimized the rapid freezing of intestinal tissue segments in large base molds placed at the surface of a slurry of dry ice in 2-MB (Figure 1B). The best success during cryosectioning and subsequent LCM was obtained by laying intestinal segm...

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Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

This method can help answer key questions about the enteric nervous system, such as the expression profile of enteric ganglia in the intestines of adult humans. The main advantage of this technique is that it can be used to obtain high yields of RNA from human enteric ganglia while maximally preserving RNA quality. Begin with intestinal tissue cut into 20 centimeter lengths.First, trim away the adipose and connective tissue. Avoid nicking the serosa, which can lead to structural damage to the myenteric plexus. Next, make a longitudinal incision along the entire length of the trimmed sample, preferably at the site of mesenteric attachment.Then, dissect away and discard the taeniae coli from the colon so the tissue can flatten out. Now, splay the intestine, mucosa side down, onto a chilled cutting board. Then, cut 1.25 centimeter wide strips along the full length of the tissue.As they are cut, transfer each piece to chilled tissue storage solution for temporary storage. Next, cut each strip into segments that are two to 1.5 centimeters long. After slicing a strip into segments, blot the segments dry.It's important that the tissue is very dry so ice crystals don't form on the tissue when it is frozen. Now, arrange the dried pieces of tissue, mucosa side up, on a cold, disposable plastic mold. Then, flatten each piece using curved forceps to remove trapped air bubbles.The tissues will expand as they are flattened. After flattening the tissue segments, assess their thicknesses prior to freezing. If needed, push the edges of the specimen inward until the intestinal sample approaches its original thickness.Now, transfer the loaded base mold onto a slurry of dry ice and 2-methylbutane. The tissue should completely freeze within 30 to 60 seconds. Then, immediately dry the bottom of the mold using cold wipes that have been kept on dry ice.Now, check the sample. Take note of any samples that do not appear flat. Next, wrap the mold in aluminum foil that has been pre-labeled and chilled before use.Then, cover the foil with a layer of plastic wrap to minimize dehydration. Keep the samples on ice until they can be stored at minus 80 degrees Celsius. After preparing the required materials, pour a three to five millimeter mound of TFM onto a large cryostat specimen holder and immediately transfer the intestinal sample, serosa side up, onto the TFM.After a few seconds at room temperature, transfer the specimen holder to dry ice and cover it with powderized dry ice. Before slicing, make sure that the TFM is below the plane of the myenteric plexus. Next, mount the specimen holder into the cryostat specimen head and align the specimen with the cryostat blade.Make the plane of the myenteric plexus parallel to the cutting blade. Now, make eight micron sections through the serosa and longitudinal muscle until reaching the myenteric plexus. To locate the myenteric plexus, mount a sample and stain it with an aqueous dye, such as 1%crystal violet.To proceed, identify the junction between the longitudinal and circular muscle layers. At the start of sectioning, the serosa will be marked by the presence of connective tissue. Some remaining mesenteric fat may also be present along the serosa.Then, the sheet of the outer longitudinal muscle layer becomes visible Once the border between the longitudinal and circular muscle layers has been reached, a portion of the enteric ganglia will be observed. In the next several sections, the myenteric plexus will become very evident with large swaths of ganglia being present within a single section. Now, collect serial sections for the laser-capture microdissection.Chill PEN slides in the cryostat for five to 10 seconds and use them to mount the sections. The best sections will have a lot of neurons and glia. Now, proceed to stain and then dehydrate the sections as described in the text protocol.In preparation, insert a cartridge of LCM caps into the LCM microscope stage. Then, load the sample slide onto the stage and acquire an overview image of the slide. Identify the desired location for LCM and load a cap onto the slide.Then, align the infrared laser and adjust the laser's power and duration for a 20 to 30 micron diameter capture spot. Next, outline the desired ganglia to be collected. Make sure to stay within the boundary of the collectible area on the cap, which is displayed as a green circle.For each of the ganglia outlined by a UV cutting trace, adjust the IR spots such that there is at least one IR spot every 100 to 500 microns to ensure the ganglia will all be collected when the cap is lifted off of the sample. Then, press the IR UV Cut button to proceed with the collection of all the marked ganglia. Once collections are completed, move the cap to a new location and repeat the collection process.This can be repeated for 60 to 80 minutes. When the LCM cap is loaded with enough ganglia, transfer it to a quality control station. If debris is present on the cap, wipe it away using a fine-tipped paint brush that has been prepared for RNA use.Use a pipette tip for any difficult to remove debris. Then, secure the cap onto a 0.5 milliliter microfuge tube filled with 230 microliters of RNA lysis buffer. Invert the cap, briefly vortex it, and then incubate the sample at room temperature for 30 minutes.Later, centrifuge the sample for five minutes at 5, 000 g or faster, and then transfer the microfuge tube to dry ice. The sample is now ready for the RNA extraction. When pulling two caps of enteric ganglia per sample, greater than one nanogram of RNA was obtained, enough for RNA-Seq, and the RNA was of excellent quality.The RNA integrity of shown sample is 8.3. Samples with integrity numbers of 7.5 and 6.9 were also collected. In total, more than 95%of all prepared samples have been good enough for RNA-Seq.The quality of the RNA-Seq data sets was assessed using a bioinformatics pipeline. The end bias indicates that the samples were successfully sequenced and only had a modest three prime end bias. A gene biotype plot shows that the sequencing results were largely consistent.Once mastered, this technique can be used to collect biological replicates of RNA from enteric ganglia of sufficient quality and quantity for RNA-Seq in as little as eight to 12 hours. While attempting this procedure, it is important that the human intestinal samples be frozen as flat as possible. This will ensure that you can collect very large cross-sections of human enteric ganglia that are quickly and easily processed with laser-capture microdissection.

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